This project is concerned with the relation of the structure to the function of dihydrofolate reductase (dihydrofolate+ NADPH + H+ - tetrahydrofolate + NADP+). Using a number of techniques of protein chemistry, the problems to be investigated include: (a) obtaining protein sequence information on a methotrexate (MTX)-insensitive enzyme from MTX-resistant L5178Y cells and from Walker 256 carcinosarcoma cells primarily by HPLC-mapping of derived peptides and amino acid analysis; (b) chemical modification of specific amino acid residues of the protein and measurements of the effect of these modifications on the substrate, cofactor and inhibitor binding and on enzymic activity; (c) equilibrium binding studies of substrate, cofactor and inhibitors using reductases from human, L5178Y and Walker 256 cells and employing fluorescence, circular dichroism and equilibrium dialysis techniques; (d) evaluation of MTX analogues as affinity and photoaffinity labels of dihydrofolate reductases; (e) examination of selected, substituted quinazolines, triazines and pteridines as enzyme inhibitors and a determination of their transport characteristics in MTX-sensitive and -resistant cells.